A major step towards understanding the physiological function of agonist-stimulated calcium entry channels in salivary gland cells requires identification of their molecular components and defining their regulation. TRPC (transient receptor potential canonical) proteins have been suggested as molecular candidates for store-operated calcium entry (SOCE) channels. SOCE is ubiquitously present in all cells and regulates a variety of cellular functions including salivary gland fluid secretion and inflammation. Our long term goal is to define the components that mediate and regulate Ca-2+ entry into salivary gland cells. Towards this goal, our studies determine cellular mechanisms which are involved in the activation and inactivation of SOCE and define the role of TRP channels in salivary gland function. These are not mutually exclusive as identification of the mechanism will facilitate identification of the channel and vice-versa. [unreadable] [unreadable] Our previous findings suggested that one or more presently known TRP proteins, including TRPC1, are molecular components of Ca-2+ entry channels in salivary gland cells. Further we showed that trafficking and assembly of store-operated and non-store-operated Ca2+ channels are key determinants of their function and regulation and that Ca-2+ entry occurs in specific microdomains where it is regulated via physical and functional association with structural and signaling proteins. In the past year we have continued to work along the same major directions. Our work has provided new information on the role and molecular composition of Ca-2+ entry channels in salivary gland cells. We have identified the translocon complex in the ER as a possible site for passive internal Ca-2+ release. In an important study we have functionally localized for the first time, the intracellular calcium stores that are coupled to store-operated calcium entry. Our studies have also revealed a novel mechanism of regulation of TRPC1-dependent store-operated calcium entry by the newly identified proteins STIM1 and Orai1. [unreadable] [unreadable] 1. Store-operated Ca-2+ entry (SOCE) is activated in response to depletion of intracellular Ca-2+ from the endoplasmic reticulum (ER). A variety of agonists stimulate SOCE via IP-3-dependent Ca-2+ depletion. SOCE is also activated by thapsigargin, an inhibitor of Ca2+ re-uptake into the ER that induces a net Ca-2+ loss from the ER by unmasking a Ca-2+ leak pathway. The molecular identity of this Ca-2+ leak channel and the physiological conditions under which such agonist-independent Ca-2+ depletion might occur remain poorly characterized. Here we report the presence of an agonist-independent mechanism for internal Ca-2+ store depletion and activation of SOCE in salivary gland cells, HSG (human submandibular gland cell line). These significant findings reveal an agonist-independent mechanism for depletion of internal Ca-2+ stores and activation of SOCE. We show that clearing of the translocon pore induces release of Ca-2+ from the internal Ca2+ store(s) and activation of SOCE. Under physiological conditions, such clearing of the translocon pore occurs following termination of protein synthesis and release of the nascent peptide. Although it is presently unclear exactly how much Ca-2+ release from the ER is required for activation of SOCE, several studies show that SOCE can be activated by submaximal depletion of internal Ca-2+ stores. Thus, depending upon the rate of protein turnover, sufficient Ca-2+ could be lost from the ER during termination of protein synthesis to activate SOCE. This link between SOCE and the protein synthesis provides a mechanism whereby the Ca-2+ in the ER can be maintained at levels optimal for protein folding and maturation in the ER. This will also be critical for protecting the cells against ER stress and activation of the unfolded protein response that result from loss of ER-Ca-2+. While it has been well established that SOCE-dependent refilling internal Ca-2+ stores provides ER-Ca-2+ for regulation of cellular functions that are mediated by agonist stimulation of PIP-2 hydrolysis, the present data demonstrate a novel agonist-independent, but physiologically critical, cellular function that is associated with ER-Ca-2+ and SOCE. [unreadable] [unreadable] 2. One of the proposed mechanisms for activation of SOCE predicts that interactions between ER and plasma membrane channel transduces the Ca-2+ status of the ER to the channel, achieving either activation or inactivation. It has been suggested that ER localized in close proximity to the plasma membrane (PM) regulates SOCE. However there are no data to directly demonstrate the location of this local ER store and whether its depletion is correlated with activation of SOCE. The recently identified SOCE-regulatory protein STIM1 is an ER-Ca2+ binding protein which relocalized to the sub-plasma membrane region when internal Ca-2+ store is depleted. This relocalization is suggested to be causal in activation of SOCE. However, it is unclear whether changes in the local Ca2+ store, i.e. in the vicinity where regulation of SOCE seems to be taking place, regulate relocation of STIM1 and activation of SOCE or whether signals from more interior regions of the cell are required. We have examined whether STIM1 regulation of SOCE is dependent on the Ca2+ in subplasma membrane ER or in more internal ER. Data obtained in this study demonstrate that mobilization of STIM1 and activation of SOCE are associated with the decrease of Ca2+ in ER which is localized within the subplasma region of the cell. Thus activation of SOCE by STIM1 is determined by the Ca2+ in the peripheral Ca2+ store where STIM1 is also localized. As long as Ca2+ in this store is below the threshold for binding to the STIM1-EF hand domain, STIM1 will be localized in punctae and SOCE will remain activated. Further studies are required to determine the exact molecular interactions involved in the generation of STIM1-containing punctae and how exactly these punctae regulate gating of the SOCE channel. [unreadable] [unreadable] 3. Despite intense focus on SOCE over the past two decades neither the mechanism(s) by which the status of Ca2+ in the ER is transmitted to the PM, to activate or inactivate SOC channels, nor the molecular components of the channels have yet been conclusively established. Recently two new proteins have emerged as candidate components of SOCE, STIM1 and Orai1. As noted above, STIM1 an EF-hand domain ER protein is shown to be essential for SOCE. The second protein, Orai1, is a four transmembrane domain protein that appears to constitute the pore-forming unit of CRAC channels which are a specific type of SOCE channels found primarily in lymphocytes and other hematopoietic cells. It has been suggested that Orai1 and STIM1 together are sufficient for the formation of this channel. We have previously reported that TRPC1 is an essential component of the SOC channels in salivary gland cells. Our data demonstrated that knockdown of TRPC1 decreases SOCE, overexpression increases the activity, while expression of TRPC1 with mutations in the proposed pore region of this protein alters the Ca2+ permeability of the channel. We have now investigated the possible role of STIM1 and Orai1 in TRPC1-dependent SOCE. This recent study reveals that all three proteins are essential for generation of TRPC1-SOC channels. Our findings show that STIM1 regulation of TRPC1-SOC is similar to its regulation of ICRAC. Based on these findings we have suggested that dynamic assembly of a TRPC1/STIM1/Orai1 complex is involved in activation of Ca2+ entry. Thus, our studies suggest a common molecular basis for channels in salivary gland cells and hematopoietic cells. Ongoing studies in our laboratory are aimed towards identifying the molecular interactions that regulate the function of this complex.